Chemotactic cytokine

ABSTRACT

A chemoattractant protein called “eotaxin” is capable of attracting eosinophils and of inducing eosinophil accumulation and/or activation in vitro and in vivo. Various types of agents that inhibit or otherwise hinder the production, release or activity of eotaxin may be used therapeutically in the treatment of asthma and other inflammatory diseases

[0001] The present invention relates to a chemotactic cytokine.

[0002] The accumulation of eosinophil leukocytes is a characteristicfeature of IgE-mediated allergic reactions such as allergic asthma,rhinitis and eczema. Eosinophil accumulation also occurs in non-allergicasthma. The immediate broncho-constriction in response to a provokingstimulus in the asthmatic involves mast cell activation and the releaseof constrictor mediators. This is followed after several hours in someindividuals by a late bronchoconstrictor response associated with amassive influx of eosinophils (1). Repeated provocation results inchronic inflammation in the airways and a marked hyper-responsiveness toconstrictor mediators. The magnitude of both the late response and thechronic hyper-responsiveness-correlates with the numbers of eosinophilspresent in the lung (2,3).

[0003] The present invention provides a chemoattractant protein capableof attracting eosinophils and of inducing eosinophil accumulation and/oractivation in vitro and in vivo. The chemoattractant protein of thepresent invention is designated “eotaxin”.

[0004] Eotaxins are proteins of the C—C branch of the platelet factor 4superfamily of chemotactic cytokines. Within the C—C branch of theplatelet factor 4 superfamily of chemotactic cytokines, or chemokines,certain members have the property of attracting eosinophils in vitro andsome may induce eosinophil accumulation in vivo. For example, thechemokines RANTES and MIP-1α attract eosinophils in vitro while MCP-1and MIP-1β do not. (“RANTES” denotes Regulated upon Activation in NormalT cells Expressed and Secreted, “MIP” denotes Macrophage InflammatoryProtein, and “MCP” denotes Monocyte Chemo-attractant Protein.)

[0005] Naturally-occurring cytokines within the platelet factor 4superfamily of chemotactic cytokines may have marked inter- the aminoacid sequence of the protein, and in the carbohydrate modifications ofthe protein, while retaining the same characteristic functionalproperties. similar variations in structure may occur in cytokinesobtained from different individuals within the same species. Manychemokines within the C—C branch of the platelet factor 4 superfamilyshow promiscuity of receptor binding, and the ability of differentchemokines to bind to the same receptor is not necessarily dependent ona high degree of homology at the amino acid level. Accordingly, bothinterspecies and intraspecies variations in protein length, amino acidsequence and carbohydrate modifications are generally to be expected foreotaxins.

[0006] The ability to attract eosinophils and to induce eosinophilaccumulation and/or activation in vitro and in vivo is a characteristicproperty of eotaxins. Furthermore, eotaxins generally show substantiallyno attractive effect for neutrophils in vivo. The eosinophilchemoattractant effect may be an inter-species effect, for example,guinea-pig eotaxin appears to be potent in inducing chemotaxis of humaneosinophils in vitro.

[0007] An eataxin may be obtained from an appropriate body fluid, forexample, from bronchoalveolar lavage fluid obtained from a human ornon-human subject, particularly an allergic subject after an allergenchallenge, either experimentally induced or naturally incurred. Othersources of eotaxins are, for example, inflammatory exudate fluids and invitro cultures of macrophages, lymphocytes, neutrophils, mast cells,airway epithelial cells, connective tissue cells, vascular endothelialcells and eosinophils themselves

[0008] For example, an eotaxin may be obtained from a sensitisedguinea-pig after allergen challenge. Guinea-pig models are useful asthey share many common features with the asthmatic response in man.Eotaxin obtainable from bronchoalveolar lavage fluid of a sensitisedguinea-pig by sequential HPLC purification generally has a molecularweight in the range of from 6-16 kDa. (As indicated above, intraspeciesmolecular weight variations of this order of magnitude are observed inmembers of the platelet factor 4 superfamily.)

[0009] The amino acid sequence of a guinea-pig eotaxin is set out inSEQ.ID. NO. 1, SEQ.ID. NO. 2 and in FIGS. 7 and 8 of the accompanyingdrawings. Other guinea-pig eotaxins will generally have at least 50%overall homology with the sequence shown in SEQ.ID. NO. 1 (FIG. 7) atthe amino acid level. The homology may be at least 60%, for example atleast 70%, for example at least 80% with the sequence set out in SEQ.ID.NO. 1 and in FIG. 7.

[0010] Percentage homology in the present case is calculated on thebasis of amino acids that are identical in corresponding positions inthe two sequences under investigation. Conservative substitutions arenot taken into account. In the calculation of percentage homology of aputative eotaxin molecule under investigation with the sequence shown inSEQ.ID. NO. 1 (FIG. 7) or with SEQ.ID. NO. 2 (FIG. 8) if the moleculeunder investigation has a different length from the ectaxin set out inSEQ.ID. NO. 1 or SEQ.ID. NO. 2 (FIG. 7 or FIG. 8), then the calculationis based on the amino acids in the portion of the molecule underinvestigation that overlaps with the sequence shown in SEQ.ID. NO. 1(FIG. 7) or SEQ.ID. NO. 2 (FIG. 8). Software packages for the alignmentof amino acid sequences and the calculation of homology are availablecommercially, for example, the “Bestfit” program available from GeneticsComputer Group Sequence Analysis Software, Madison, Wis., U.S.A.

[0011] Unless specified otherwise, the specific values of percentagehomology between eotaxin and other chemotactic cytokines given in thepresent specification have been calculated on the basis of the eotaxinset out in SEQ.ID. NO. 1 (FIG. 7).

[0012] As indicated above, eotaxins obtainable from species other thanguinea-pigs, for example humans, will exhibit inter-species differencesof the type demonstrated by other members of the C—C branch of theplatelet factor 4 superfamily of chemokines, for example, differences inprotein length, amino acid sequence and carbohydrate modifications Theremay, for example, be variations in the C- and/or N-terminal residues.For example, it is expected that the molecular weight of an eotaxin froma species other than guinea-pig will generally fall within the range offrom 6 kDa to 16 kDa, but in some cases an eotaxin may have a molecularweight less than 6 kDa or more than 16 kDa.

[0013] Similarly, it is expected that in general an eotaxin from aspecies other than guinea-pig will have at least 40% overall homologywith the sequence set out in SEQ.ID. NO. 1 and in FIG. 7 The homologymay be at least 50%, for example at least 60%, for example at least 70%,for example at least 80% with the sequence set out in SEQ.ID. NO. 1 andin FIG. 7 There may, however, be eotaxins from species other thanguinea-pigs that have less than 40% homology with SEQ.ID. NO. 1 (FIG.7).

[0014] Eotaxins may be identified by any one or more of thecharacteristics set out above, in particular by their ability to attractand/or active eosinophils in vitro and cause their accumulation and/oractivation in vivo. A characteristic that assists the identification ofa molecule as an eotaxin is the lack of attractive effect onneutrophils.

[0015] The present invention provides a method of determining theability of a substance to induce eosinophil accumulation and/oractivation in vivo, that is to say, a method for testing putativeeotaxins, which comprises administering the substance, generallyintradermally, to a test animal previously treated with labelled,for-example ¹¹¹In-labelled, eosinophils and subsequently determining thenumber of labelled eosinophils at a skin site.

[0016] One in vitro method that may be used to test a putative eotaxinfor the ability to attract and/or activate eosinophils in vitro is theability of the substance to increase eosinophil intracellular calciumlevels. Other general methods for determining chemotactic activity invitro may be used to test putative eotaxins in vitro.

[0017] Confirmation that an eosinophil attractant is an ectaxin may alsobe made by consideration of sequence homology of that protein with thesequence set out in SEQ.ID. NO. 1 (FIG. 7) and/or with the sequence setin SEQ.ID. NO. 2 (FIG. 8) and/or by consideration of the structuralrelationship between the protein and the guinea-pig eotaxin.

[0018] As mentioned above, RANTES and MIP-1α are both eosinophilactivators. Eotaxin has functional similarities but low structuralhomology with RANTES and MIP-1α (31% homology with MIP-1α at the aminoacid level calculated on the basis of SEQ.ID. No. 1 (FIG. 7); 32%homology when calculated on the basis of the overlapping sequences and26% homology with RANTES at the amino acid level calculated on the basisof SEQ.ID. No. 1 (FIG. 7); 27% homology when calculated on the basis ofthe overlapping sequences). An eotaxin can be distinguished from RANTESand MIP-1α not only by the degree of homology but also by the overalldifferences in sequence and structure.

[0019] In addition to full-length eotaxin molecules, the presentinvention also provides molecules that comprise less than a, full lengtheotaxin sequence. Such molecules (called “fragments” herein) may bepolypeptides or peptides. For use as an eotaxin substitute, a fragmentshould retain one or more of the biological activities of the parentmolecule.

[0020] Eosinophils contain an armoury of chemicals necessary for killingparasites. These chemicals have been implicated in the damage to airwayepithelium that occurs in asthma and may relate to the observed changesin airway function (26,27). From our studies we suggest that eotaxinsshould be considered as important mediators of eosinophil accumulationin vivo. Macrophages, lymphocytes, neutrophils, mast cells, airwayepithelial cells, connective tissue cells, vascular endothelial cellsand eosinophils themselves are likely candidates as the source of theeosinophil chemoattractant activity generated in the lung. Platelets mayalso have a role as it has been shown that they can release C—Cchemokines (22). Further, an early platelet deposition may be involvedin the subsequent eosinophil accumulation in vivo (28,29) and there isevidence that platelet-activating factor induces the synthesis of anunidentified eosinophil chemoattractant in vivo (30). In this respect,it is of interest that platelet-derived growth factor can induce geneexpression of C—C chemokines in fibroblasts (31). Furthermore, the C—Cchemokines have been implicated in wound healing (18). This may beimportant in the sub-epithelial basement membrane fibrosis that is aprominent feature of the asthmatic lung. Thus, eotaxins may be involvedin both eosinophil accumulation and in chronic structural changes in thelung.

[0021] Eotaxins may have an important role in asthma and in otherdiseases having an inflammatory component where eosinophil accumulationand/or activation is a prominent feature, for example, rhinitis andeczema, especially allergic eczema. Accordingly, agents that inhibit orotherwise hinder the production, release or action of eotaxins havepotential as selective therapeutic agents. Such agents and theirtherapeutic use are part of the present invention.

[0022] Such agents include inhibitors that affect the interaction of aneotaxin with eotaxin receptors, for example, by binding to an eotaxin orto an eotaxin receptor. An example of such an inhibitor is receptorsthemselves which, on administration, can bind an eotaxin and prevent itsinteraction with naturally-occurring receptors. Such inhibitoryreceptors may be soluble or insoluble. Receptors which are not involvedin cell activation may be bound to, or induced on, cells. Such receptorsmay also be used to remove endogenous eotaxin.

[0023] Further examples of agents that affect the interaction ofeotaxins with eotaxin receptors are receptor antagonists, andantibodies, both antibodies directed against (capable of binding with)an eotaxin and antibodies directed against an eotaxin receptor,especially monoclonal antibodies. Any other agent that inhibits orotherwise hinders the binding of an eotaxin to an eotaxin receptor alsohas therapeutic potential, for example, any other agent that binds to aneotaxin or to an eotaxin receptor. Further agents that have therapeuticpotential are those that prevent or reduce activation of eotaxinreceptors.

[0024] Further agents that inhibit or otherwise hinder the action ofeotaxins are those that change the structure of an eotaxin such that itis no longer able to bind to an eotaxin receptor, for example, an enzymeor other agent that degrades eotaxin specifically.

[0025] Receptor promiscuity is common among chemokines, so although itis essential that a receptor is capable of binding an eotaxin, thereceptor need not necessarily be eotaxin-specific. For example, areceptor may bind MIP-1α, RANTES and/or other eosinophil attractantchemokines as well as an eotaxin.

[0026] As indicated above, possibilities for therapeutic interventioninclude the use of a receptor to which an eotaxin binds, especially asoluble receptor. It may be advantageous to use an eotaxin-specificreceptor. Further possibilities for therapeutic intervention includereceptor antagonists, for example, based on 3-dimensional structures orthe amino acid sequences of eotaxins and/or of eotaxin receptors, andagents found to inhibit eotaxin or other agonists binding to oractivating eotaxin receptors. For example, a receptor antagonist or anagonist inhibitor may be a polypeptide in which the sequence of afull-length naturally-occurring eotaxin has been modified, for example,by amino acid substitution, or may be a fragment of an eotaxin (that isto say, a polypeptide or small peptide comprising part of the amino acidsequence of a naturally-occurring eotaxin), or a modified fragment of aneotaxin, for example, modified by amino acid substitution.

[0027] Furthermore, knowledge of the sequence and/or structure ofeotaxins either alone or in combination with knowledge of the sequenceand/or structure of other chemokines that bind to the same receptor(s)as eotaxins, provides useful information for the design of therapeuticagents.

[0028] Agents that prevent or inhibit eotaxin synthesis or release mayalso be used therapeutically. Such agents and their use are also part ofthe present invention.

[0029] All inhibitors of eotaxin activity, synthesis and release,including soluble receptors, antibodies, antagonists and inhibitors ofagonist binding, and their use are part of the present invention.

[0030] The present invention accordingly provides an agent that inhibitsor otherwise hinders the production, release or action of an eotaxin,especially an agent as described above, for use as a medicament. Theinvention also provides the use of an agent that inhibits or otherwisehinders the production, release or action of an eotaxin, especially anagent as described above, in the manufacture of a medicament for thetreatment of asthma or another disease having an inflammatory component,particularly with accumulation of eosinophils, for example, rhinitis oreczema, especially allergic eczema.

[0031] The use of the structural and sequence information relating toeotaxins in the design of therapeutically and diagnostically usefulagents, for example, in computer-aided design based on the threedimensional structure of eotaxins is part of the present invention.

[0032] Putative inhibitors of eotaxin activity may be screened using invivo and in vitro assays based on inhibition of chemoattraction and/oraccumulation and/or activation of eosinophils by eotaxins. Some generalmethods for testing the activity of a compound for an inhibitory effecton the activity of a chemoattractant cytokine in vitro are known. Suchassays may be used to determine the inhibitory action of a putativeinhibitor on in vitro effects induced in eosinophils by eotaxins.

[0033] Assays that are suitable for screening putative eotaxininhibitors include, for example, inhibition in vitro of elevation ofintracellular calcium levels induced in cells by eotaxin. The method ofthe present invention for determining the ability of a substance toinduce eosinophil accumulation and/or activation in vivo, that is tosay, a method for testing putative eotaxins, may also be used todetermine the ability of a substance to inhibit eosinophil accumulationand/or activation induced in vivo by an eotaxin: an animal is pretreatedwith labelled eosinophils, an eotaxin and a putative inhibitor areadministered, and the number of labelled eosinophils at a skin site aredetermined subsequently. The eotaxin is generally administeredintradermally, and the putative inhibitor may be administered by thesame route or by a different route, for example systemically.

[0034] Examples of in vitro and in vivo assays both for thedetermination of eotaxin activity and for the determination of eotaxininhibitory activity are described herein. For example, Example 1 gives adetailed protocol for the in vivo assay of the present invention, andExample 4 gives detailed protocols of various assays. The assaysdescribed herein may be used as such, or may be modified as required.Assays may be used alone or in combination to establish eotaxin andeotaxin-inhibitory activity. A putative inhibitors may be any of thetypes of molecules described above, including receptors, for example,soluble receptors, antibodies, and antagonists and inhibitors of agonistbinding. Methods for testing putative inhibitors of eotaxins are alsopart of the present invention.

[0035] A further aspect of the present invention is a pharmaceuticalpreparation comprising, as active ingredient, an agent that inhibits orotherwise hinders the production, release or action of an eotaxin, inadmixture or in conjunction with a pharmaceutically suitable carrier.Such agents are described above and include, for example, an inhibitorof eotaxin synthesis or release, a soluble eotaxin receptor, an eotaxinreceptor antagonist or an inhibitor of an eataxin receptor agonist, anantibody against eotaxin or an antibody against an eotaxin receptor.

[0036] The invention further provides a method of treating asthma andother inflammatory diseases, comprising the administration of aneffective amount of an agent that inhibits or otherwise hinders theproduction, release or action of an eotaxin. The agent may be asdescribed above, for example, an inhibitor of eotaxin synthesis orrelease, a soluble eotaxin receptor, an eotaxin receptor antagonist oran inhibitor of an eotaxin receptor agonist, or an antibody againsteotaxin or against an eotaxin receptor.

[0037] The present invention also provides assays for eotaxins and foranti-eotaxin antibodies, especially immunoassays and in particularELISAs (enzyme-linked immunosorbent assays). The invention provides, forexample, an immunoassay for an antigen, characterised in that theantigen is an eotaxin, and also provides an immunoassay for an antibody,characterised in that the antibody is an anti-eotaxin antibody. Theinvention also provides assays for eotaxins that are analogous toimmunoassays for eotaxins but that use a specific-binding partner otherthan an antibody. In such specific-binding partner assays an eotaxinreceptor may be used instead of an anti-eotaxin antibody.

[0038] In an immunoassay, an anti-eotaxin antibody may, for example, becoated on a solid surface to enable capture and hence detection ofeotaxin An anti-eotaxin antibody may be used in an assay for thedetection of antibodies to eotaxin, for example, in a competitiveantibody assay. A labelled eotaxin or a derivative thereof, for example,a recombinant eotaxin or a synthetic peptide comprising part of theamino acid sequence of an eotaxin may be used in a competitive antigenassay for eotaxin or may be used to coat a solid surface in a captureassay for antibodies to eotaxin. The many different types of assayformat are well described in the literature of the art, see for example“ELISA and other Solid Phase Immunoassays, Theoretical and PracticalAspects” Eds Kemeny D.M. & Challacombe S. J., John Wiley, 1988. (36).Assays using an eotaxin receptor instead of an anti-eotaxin antibody maybe carried out analogously.

[0039] The present invention provides a process for the production of aneotaxin, which comprises obtaining bronchoalveolar lavage fluid obtainedfrom a human or non-human animal challenged with a provoking stimulus,for example, from a human suffering from allergic or non-allergicasthma, or other lung disease, or a guinea-pig sensitised with a foreignprotein, and isolating a fraction showing eosinophil chemoattractantactivity. One method of isolating an eotaxin-containing fraction ofbronchoalveolar lavage fluid is sequential cation exchange, sizeexclusion and reversed phase HPLC systems. The desired fractiongenerally contains a polypeptide having a molecular weight in the rangefrom 6-16 kDa. Purity may be verified by SDS-PAGE. If desired, theauthenticity of the substance obtained may be determined by comparisonof the amino acid sequence thereof with the amino acid sequence set outin SEQ.ID. NO. 1 or SEQ.ID. NO. 2 (FIG. 7 or FIG. 8).

[0040] Eotaxins may be obtained according to the above procedure fromother sources, for example, from inflammatory exudate fluids, or from invitro cultures of macrophages, lymphocytes, neutrophils, mast cells,airway epithelial cells, connective tissue cells, vascular endothelialcells and eosinophils themselves.

[0041] Alternatively, a full-length eotaxin, or a part (fragment) of aneotaxin, for example, a polypeptide or peptide fragment, may be producedby chemical synthesis, for example, according to the Merryfieldtechnique. A further method for producing a full-length eotaxin or apart thereof is by recombinant DNA technology. All methods of producingeotaxin are part of the present invention.

[0042] To produce a full-length eotaxin polypeptide or a polypeptide (orpeptide) fragment by recombinant DNA technology, a nucleic acid sequenceencoding the polypeptide is inserted into an expression vector under thecontrol of appropriate control sequences. A recombinant polypeptide maythen be expressed using a prokaryotic expression system, for example, inE. coli, or using a eukaryotic cell system, in which case the resultingpolypeptide may be glycosylated. Such techniques are standard see, forexample Sambrook, J., Fritisch, E. F. and Maniatis T., Molecular CloningA Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989 (37).

[0043] A nucleic acid encoding all or part of an eotaxin polypeptide maybe obtained by screening a library prepared from suitable cells, forexample, cells from allergen-challenged guinea-pig or human lung.Screening may be carried out using a probe comprising sequencescharacteristic of an eotaxin, in particular, sequences that distinguishthe eotaxin from other related cytokines, for example, RANTES andMIP-1α. It may be preferable to use a long probe, for example, a probecomprising a nucleic acid sequence encoding a full-length eotaxinpolypeptide.

[0044] Alternatively, the polypeptide sequence of an eotaxin may be usedto design oligonucleotide primers, for example, degenerate primers.Primers may, for example, comprise bases less specific in theirinteraction than the naturally-occurring bases, for example, inosine maybe used. Examples of primers are the sense sequence

[0045] 5′ TGC TGT TTC CGI GTI ACT AAC AAA (SEQ.ID.NO. 3) based on theamino acid sequence CCFRVTNK, and the anti-sense sequence

[0046] 5′ CAT CTT GTC IGG CTT TAT TTC (SEQ.ID. NO. 4)

[0047] based on the amino acid sequence EIKPDKM. Such primers may beused for amplification of reverse transcribed mRNA by the polymerasechain reaction. This provides cDNA probes for screening libraries, forexample, as described above, to isolate full length eotaxin clones.Primers may include codons chosen on the basis of known speciespreference.

[0048] As indicated above, derivatives of naturally occurring eotaxinsare also part of the present invention. Such derivatives includepolypeptides that have one or more of the following modificationsrelative to a naturally-occurring eotaxin:

[0049] (i) elongation or shortening at the C-terminus;

[0050] (ii) elongation or shortening at the N-terminus;

[0051] (iii) deletion and/or insertion of internal sequences; (iv) aminoacid substitutions, for example at the C- and/or N-terminus; and

[0052] (v) a different pattern of glycosylation. (There is, for example,a potential O-glycosylation site at residue 70.)

[0053] Such derivatives may function as agonists for structure/activityrelationship studies or as receptor antagonists.

[0054] Anti-eotaxin antibodies and anti-eotaxin receptor-antibodies,both polyclonal and monoclonal, may be produced according to standardtechniques, for example, Kohler & Milstein (38). A full-length eotaxinmay be used as antigen, or it may be preferred to use a fragment of aneotaxin in order to produce an antibody to a specific antigenicdeterminant A naturally occurring eotaxin may used as antigen.Alternatively, a recombinant or synthetic eotaxin or eotaxin polypeptideor peptide may be used.

[0055] FIGS. 1 to 18 of the accompanying drawings illustrate the presentinvention. The following is a brief description of the Figures. A moredetailed description of the Figures is given below and in the Examplessection of this specification.

[0056]FIG. 1: Protocol for the generation and assay of eosinophilchemoattractant activity in vivo.

[0057]FIG. 2: Time course of generation of eosinophil chemoattractantactivity in lungs of sensitized guinea-pigs after antigen challenge.

[0058]FIGS. 3, 4 and 5 all relate to the purification of eotaxin frombronchoalveolar lavage (BAL) fluids:

[0059]FIG. 3: Final reversed phase HPLC profile showing absorbance at214 nm and the acetonitrile gradient.

[0060]FIG. 4: Measurement of ¹¹¹In-eosinophil chemoattractant activityof the fractions obtained on HPLC chromatography.

[0061]FIG. 5: Measurement of ¹¹¹In-neutrophil chemoattractant activity;lack of significant activity observed.

[0062]FIG. 6: SDS-PAGE analysis of C18 reversed phase HPLC fractions50-56.

[0063]FIGS. 7 and 8: Amino acid sequences of an isolated guinea-pigeotaxin.

[0064]FIG. 9: Comparison of the eotaxin sequence of FIG. 7 with humanMCP-1, MCP-2, MCP-3 (21), guinea-pig MCP-1 (24), human MIP-1α, MIP-1βand RANTES (18) showing conserved residues (shaded).

[0065]FIG. 10: Comparison of ¹¹¹In-eosinophil accumulation in vivo usingguinea-pig eotaxin and the recombinant human proteins RANTES, MIP-1α andMCP-1.

[0066]FIG. 11 (Inset): Inhibition of the binding of ¹²⁵I-RANTES toguinea-pig eosinophils in vitro induced by eotaxin and RANTES but not bya member of the C—X—C branch of chemokines, IL-8.

[0067]FIGS. 12, 13 and 14 illustrate the potential of blocking theresponse to guinea-pig eotaxin. This makes use of the fact that humanRANTES, whilst competing with eotaxin for binding sites, does notactivate guinea-pig eosinophils.

[0068]FIG. 12: Elevation of intracellular calcium levels in humaneosinophils in vitro induced by eotaxin, RANTES and, at highconcentration only, MCP-1.

[0069]FIG. 13: Elevation of intracellular calcium levels in guinea-pigeosinophils in vitro induced by eotaxin, but not human RANTES and MCP-1.

[0070]FIG. 14: Representative traces showing inhibition of guinea-pigeotaxin-induced elevation of intracellular calcium levels in guinea-pigeosinophils in vitro by the previous exposure of the same cells to humanRANTES. Inset:

[0071]FIG. 15: Values (mean±SEM) for four separate eosinophilpreparations.

[0072]FIG. 16: Dose response curve showing inhibition of guinea-pigeotaxin-induced elevation of intracellular calcium levels in guinea-pigeosinophils in vitro by the previous exposure of the same cells to humanRANTES.

[0073]FIG. 17: Histogram showing comparison of in vivo response ofguinea-pig eosinophils to eotaxin, RANTES and RANTES plus eotaxinco-injected intradermally.

[0074]FIG. 18: Histogram showing number of eosinophils and neutrophilsin bronchoalveolar lavage fluid (BAL) after administration of guinea-pigeotaxin as an aerosol to guinea-pig airways.

[0075] As in allergic asthmatic patients, exposure of sensitisedguinea-pigs to aerosolised antigen results in an immediate phase ofbronchoconstriction with associated mast cell degranulation followed, insome individuals, by a late phase of bronchoconstriction and airwayhyper-responsiveness (4-7). Although clearly no one model mimics all thefeatures of the human disease, the guinea-pig model shares commonfeatures with the asthmatic response in man and has been extensivelyused to investigate possible mechanisms (7). In particular, in bothguinea-pig and man, the immediate response to allergen triggers thesubsequent accumulation in the lung of high numbers of eosinophils.Experiments were designed to detect the appearance in the lung ofchemoattractants that may be responsible for the accumulation ofeosinophils. A strategy was employed that has previously been applied tothe identification of neutrophil chemoattractants in inflammatoryexudates (8-10).

[0076] Guinea-pigs were sensitised with intraperitoneal ovalbumin on day1 followed by a short exposure of their lungs to aerosolised ovalbuminat day 8. On day 15-21 animals were challenged with aerosolisedovalbumin and killed at different intervals using a barbiturateoverdose. Immediately after death the airways were lavaged with saline.The broncho-alveolar lavage (BAL) fluid was centrifuged to remove cells,and supernatants were either stored at −20° C. for assay or subjected topurification. Eosinophil chemoattractant activity in BAL fluid samplesor HPLC fractions was tested by injecting them intradermally into assayguinea-pigs previously given intravenous injections of ¹¹¹In-eosinophils(11,12). After a 2 or 4 h interval, assay animals were killed and thepunched out skin sites were counted in a gamma-counter (FIG. 1).

[0077]FIG. 2 shows the time-course of appearance of eosinophilchemoattractant activity in BAL fluid. Significant activity was observed30 min after antigen challenge. Activity increased up to 3 h, remainedhigh at 6 h, but was not significant in 24 h samples. Control samples(BAL fluid from sham-sensitised/challenged or sensitised/sham-challengedguinea-pigs) taken at 3 h had no significant activity.

[0078] Eosinophil chemoattractant activity, which we termed eotaxin, waspurified from 3 h BAL fluid by sequential cation exchange, sizeexclusion and reversed phase HPLC using the in vivo ¹¹¹In-eosinophilaccumulation assay to measure the activity of fractions throughout.Eotaxin eluted as a single discreet peak of bioactivity from both thecation exchange and the size exclusion steps, indicating a stronglycationic protein of 7-14 kDa. Reversed phase chromatography separatedeosinophil chemoattractant activity into two peaks (fractions 51+52 andfraction 54), which were associated with discreet peaks of proteinabsorbance (FIGS. 3 and 4). Selectivity for eosinophils was shown by thelack of significant neutrophil chemoattractant activity in thesefractions as measured by the accumulation of ¹¹¹In-neutrophils in theskin assay (FIG. 5). Histological examination of skin injected witheotaxin (2 pmol) demonstrated eosinophil accumulation at 4 and 24 h,particularly around small blood vessels (haematoxylin and eosin stains).

[0079] SDS-PAGE analysis revealed a single protein band in each offractions 51, 52 and 54 (FIG. 6). The protein in fractions 51 and 52 wasslightly larger than that in fraction 54. This was confirmed by massanalysis in which the major signals were at approximately 8.81 and 8.38kDa respectively (see Example 3). N-terminal sequencing of fractions 51,52 and 54 revealed identical amino acid sequences (see FIG. 7). TheN-terminal 37 residue sequence of eotaxin as set out in SEQ.ID. No. 1and FIG. 7 shows 57% homology with human monocyte chemotactic protein(MCP-1 (13), otherwise known as MCAF (14) and JE (15)). Tryptic peptidesof fraction 54 were also sequenced and readily aligned by comparisonwith human MCP-1 to give the virtually complete sequence of eotaxin withan overall homology of 53%, see FIG. 7. It is likely that the variationsin molecular mass reflect differential glycosylation as the four masssignals obtained (two major and two minor, see Example 3) are alldifferent from each other by multiples of approximately 220 mass units.The sequence contains no N-glycosylation sites, but a potentialO-glycosylation site at position 70 has been identified (see Example 3).Human MCP-1 also exhibits heterogeneity on SDS-PAGE due to differencesin the O-linked carbohydrate modification (16).

[0080] The platelet factor 4 superfamily of chemotactic cytokines, orchemokines, is characterised by four conserved cysteines. The relativeposition of the two N-terminal cysteines allows the subdivision of thissuperfamily into the C—X—C chemokines (eg. IL-8 (17)) which arepredominantly neutrophil chemoattractants and the C—C chemokines (eg.MCP-1, RANTES, MIP-1α and MIP-1β (18)) which are chemotactic forleukocytes other than neutrophils. Eotaxin is a member of the C—C branchof chemokines. The greatest homology (53%) is with human MCP-1 which, inthe limited in vitro studies to date, has been reported to be inactiveon human eosinophils (19,20) and with the recently described human MCP-2(54%) and MCP-3 (51% homology calculated on the basis of SEQ.ID. No. 1(FIG. 7); 54% homology when calculated on the basis of the overlappingsequences) (21). Homology with other human C—C chemokines (FIG. 9) is:MIP-1β (37% calculated on the basis of SEQ.ID. No. 1 (FIG. 7); 39% whencalculated on the basis of the overlapping sequences), MIP-1α (31%calculated on the basis of SEQ.ID. No. 1 (FIG. 7); 32% when calculatedon the basis of the overlapping sequences) and RANTES (26% calculated onthe basis of SEQ.ID. No. 1 (FIG. 7); 27% when calculated on the basis ofthe overlapping sequences). The latter two proteins have recently beenshown to be potent eosinophil activators in vitro (20,22) whereas MIP-1βactivates lymphocytes in vitro (23) but apparently not eosinophils (20).Eotaxin shows the greatest structural homology with human MCP-1, MCP-2and MCP-3. Eotaxin has functional similarities, but relatively lowhomology, when compared with RANTES and MIP-1α. Eotaxin is clearly adistinct molecule from guinea-pig MCP-1; the latter has recently beencloned (24) and it has only a 43% homology with the eotaxin sequence,see FIG. 9. Guinea-pig MCP-1 was shown to be chemotactic for monocytesbut was not tested on eosinophils (24). Interestingly, eotaxin has a 41%homology with a C—C protein whose gene is expressed in mouse mast cellsand upregulated 2h after the interaction between IgE and antigen (25).No functional activity has been reported for this protein but it isdistinct (51% homology) from mouse MCP-1/JE (25).

[0081] Guinea-pig eotaxin was very potent in inducing eosinophilaccumulation in vivo: 1-2 pmol/skin site giving a 730±140% response(mean±s.e.m., n=18 animals) compared with saline-injected sites.Further, marked eosinophil accumulation was seen within 30 minutes ofintradermal injection (FIG. 10). This is supported by experiments invitro. The effect of ectaxin on eosinophils in vitro was shown by (i)inhibition of binding of ¹²⁵I-RANTES to guinea-pig cells (FIG. 11), (ii)elevation of cytoplasmic calcium in human and guinea-pig cells (FIGS. 12and 13) and (iii) chemotaxis of human eosinophils in a Boyden chambersystem: the chemotactic responses to eotaxin and RANTES were of similarmagnitude over the range 0.1-3.0 nM. In contrast to eotaxin, humanrecombinant MCP-1 and RANTES did not induce guinea-pig eosinophilresponses in vivo or in vitro (FIGS. 10 and 13). This may reflect aspecies difference although RANTES did bind to guinea-pig eosinophilswithout inducing activation (FIG. 11, inset in FIG. 10). Eotaxin has asimilar potency to RANTES on human eosinophils whereas MCP-1 is eitherinactive (20) or active only at high doses (FIG. 12). Thus, eotaxin hasa potent direct effect on both human and guinea-pig eosinophils.

[0082] Responses of guinea-pig eosinophils to guinea-pig eotaxin invitro and in vivo can be inhibited by human RANTES. FIGS. 14, 15 and 16show the response of FURA-2-loaded guinea-pig eosinophils to guinea-pigeotaxin before and after the addition of human RANTES. Theeotaxin-induced increase in intracellular calcium concentration isreduced to a substantial degree when the cells are first exposed tohuman RANTES, which itself fails to induce a response. FIG. 17 showsthat human RANTES, when coinjected with the eotaxin, reduces theaccumulation of guinea-pig eosinophils induced by eotaxin in guinea-pigskin in vivo. The results support our observations that eotaxin exhibitscompetitive binding with radiolabelled human RANTES on guinea-pigeosinophils and that eotaxin is a potent functional stimulant whereasRANTES is not. Accordingly, RANTES appears to act as a receptorantagonist for eotaxin on guinea-pig eosinophils.

[0083]FIG. 18 shows that eosinophils accumulate in guinea-pig airways invivo within 24 hours of the administration of aerosolized guinea-pigeotaxin, whereas substantially no accumulation of neutrophils isobserved.

[0084] The following Examples illustrate the present invention.

EXAMPLE 1

[0085] Production and Testing of Bronchoalveolar Lavage Fluid

[0086] METHODS:

[0087] Male Dunkin Hartley guinea-pigs (300-400 g) were sensitised withintraperitoneal ovalbumin (OA, 1 mg) on day 1 followed by exposure toaerosolised antigen (2% OA for 5min using an ultrasonic nebuliser) onday 8 (6). On day 15-21, animals were pretreated with an antihistamineto prevent acute fatality (pyrilamine, long kg-⁻¹, i.p.) and challengedby exposure to aerosolised antigen (1% OA for 20 min). At differenttimes after antigen challenge animals were treated with atropine (0.06mg kg⁻¹, i.p.) to prevent bronchoconstriction and killed with abarbiturate overdose. Bronchoalveolar lavage was performed with 4 mlsaline. Samples were centrifuged to remove cells and the supernatantstored at −20° C. prior to assay. BAL samples were assayed byintradermal injection (0.1 ml) into guinea-pigs previously givenintravenous injections of 5×10⁶ ¹¹¹In-eosinophils (elicited in donoranimals by repeated intraperitoneal injections of horse serum andpurified on discontinuous Percoll gradients, >94% purity) (11,12). After4 hours, assay animals were killed and the skin sites punched out forgamma-counting. Data are presented as the mean±s.e.m. and were tested byone way analysis of variance. A p value of <0.05 was consideredstatistically significant.

[0088] RESULTS:

[0089]FIG. 1 shows, schematically, procedures for the generation andmeasurement of eosinophil chemoattractant activity in vivo. FIG. 2illustrates the time course of generation of eosinophil chemoattractantactivity in lungs of sensitised guinea-pigs after antigen challenge(filled squares, n=4-10). Activity was measured in an in vivo skin assayof ¹¹¹In-eosinophil accumulation in unsensitised guinea-pigs (3 testanimals per BAL sample). Significant activity was seen inbronchoalveolar lavage samples taken 0.5, 1.5, 3 and 6 h after antigenchallenge (compared to responses to intradermal saline, shown as thedotted line). No significant activity was observed in 24 h samples. Noactivity was seen in lavage samples obtained 3 h after sham (saline)challenge of sensitised animals (filled circles, n=5) or antigenchallenge of sham (saline) sensitised animals (filled diamonds, n=5).

EXAMPLE 2

[0090] Purification of Eotaxin from Bronchoalveolar Lavage Fluids

[0091] METHODS:

[0092] Bronchoalveolar lavage (BAL) fluid collected from 25 sensitisedguinea-pigs (each lavaged with 4 ml, followed by 2×10 ml, saline) 3 hafter antigen challenge (1% OA, 5 min exposure) was applied to a cationexchange HPLC column (Ultropac TSK535CM, 7.5×150 mm). Activity waseluted at approx 1.4M ammonium acetate, pH 5.5, and lyophilised for sizeexclusion HPLC (Ultropac TSK columns SWP, 7.5×75 mm, G4000SW, 7.5×600mm, and G2000SW, 7.5×600 mm, in series, equilibrated in 0.08% TFA).Activity eluted at approx 7-14 kDa. This was applied to a wide pore (300A) Vydac C18 reversed phase HPLC column (4×250 mm) in 0.08% TFA, elutedwith a linear gradient of acetonitrile (0-80% ACN in 0.08% TFA, over 80mins, at 1 ml min⁻¹) and 0.5 min fractions were collected. Aliquots (4%)of each fraction were lyophilised in the presence of carrier protein(ESA, <0.1 ng endotoxin mg⁻¹) and redissolved in 0.8 ml saline fortesting in the skin bioassays of ¹¹¹In-eosinophil and ¹¹¹In-neutrophilaccumulation over 2 hours (n=4). Eosinophils (99%, 0.5% neutrophils)were elicited with 10 repeated intra-peritoneal injections of horseserum (11,12) and neutrophils (99.4%, 0.6% eosinophils) were elicitedwith a single intraperitoneal injection of casein (5% w/v, 15 ml) 17 hprior to purification on discontinuous Percoll gradients and labellingwith ¹¹¹In.

[0093] RESULTS:

[0094] The results are presented in FIGS. 3, 4 and 5. FIG. 3 shows thefinal reversed phase HPLC profile showing absorbance at 214nn and theacetonitrile gradient. FIG. 4 shows that eosinophil chemoattractantactivity was seen in 2 peaks, corresponding to fractions 51+52 andfraction 54, which corresponded to discreet peaks of absorbance. FIG. 5shows that no significant neutrophil chemoattractant activity wasdetected in these fractions. In contrast, guinea-pig C5a des Arg (30%zymosan-activated plasma (11), approx 10 pmol/site) induced theaccumulation of both ¹¹¹In-eosinophils (5211±893) and ¹¹¹-neutrophils(9872±473). Fractions 50, 53, 55 and 56 consistently gave little or noactivity in the guinea-pig skin bioassays of leukocyte accumulation. Nosignificant protein absorbance was detected in the remainder of thegradient (up to 80% acetonitrile).

EXAMPLE 3

[0095] Purity, Mass Analysis and Protein Sequence of Eotaxin

[0096] METHODS:

[0097] 2% aliquots of each fraction were lyophilised, redissolved in 10μl SDS buffer, heated (95° C., 5 min) and ^(0.3) μl run on 8-25%gradient gels in a Pharmacia Phast System. Gels were visualised withsilver staining. Mass analysis was performed on fractions 51, 52 and 54using a Finnigan MAT Lasermat with a-cyano-4-hydroxycynnamic acid andsinapinic acid matrices. Mass measurements were calibrated internallyusing protein standards. 5% aliquots of each bioactive fraction (51, 52and 54) were applied directly to automated N-terminal sequence analysisusing fast cycles on an Applied Biosystens 477A containing amicrocartridge essentially as described (32). The amino-terminal 37, 35and 29 residues were obtained for fractions 51, 52 and 54 respectively.No differences between corresponding positions were found. The apparentinitial yields of these three analyses were all approximately 7-8 pmol.Thus fractions 51, 52 and 54 contained approx 200 pmol each, assuming70-80% sequencing yields. Gaps were found at positions 8,9 and 33,consistent with the presence of cysteine residues at these positions.

[0098] Approximately 30 pmol of fraction 54 was reduced and alkylated bysequential treatment with 1 mM dithiothreitol for 5 min at 50° C. andthen 10 mM acrylamide for 30 min at 37° C. before digestion withalkylated trypsin (Promega) in 20 mM Tris/HCl, pH 8.8, containing 0.5%Thesit. Peptides were separated using a Reliasil C18 (300 Å, 5 μm)column (1×150 mm) developed with a linear acetonitrile concentrationgradient in 0.08% trifluoroacetic acid at 50 μl/min on a Microm HPLCsystem. Purified peptides were subjected to N-terminal sequence analysisas above, but all four cysteine residues were positively identified asthe PTH-cys-S-β-propionamide derivative (33). Position 70 gave no PTHderivative in peptides T6 and T7 and is a probable position ofO-glycosylation.

[0099] RESULTS:

[0100] The results are presented in FIGS. 6, 7, 8 and 9. FIG. 6 is aphotograph of the SDS-PAGE gel. For reference, human IL-8 (72 aminoacids, approx 8 kDa) was run in lanes A, B and C (12, 2.4 and 0.5 ng/0.3μl lane respectively). Laser desorption time of flight mass analysisgave signals at 8.81 kDa (major) and 9.03 kDa (minor) for each offractions 51 and 52 Fraction 54 gave signals at 8.38 kDa (major) and8.15 kDa (minor).

[0101]FIG. 7 and SEQ.ID. NO. 1 show the amino acid sequence of eotaxin,which was determined by sequencing the intact molecule as well aspeptides derived from digestion with trypsin (T). N-terminal analysesshowed the highest homology with human MCP-1 (57%) and the trypticpeptides were readily aligned by comparison with the human MCP-1sequence. FIG. 8 shows the amino acids sequence as confirmed by nucleicacid sequencing. FIG. 9 is a comparison of the eotaxin sequence withhuman MCP-1, MCP-2, MCP-3 (21), guinea-pig MCP-1 (24), human MIP-1α,MIP-1β and RANTES (18) showing conserved residues (shaded).

EXAMPLE 4

[0102] In vitro and in vivo Testing of Eotaxin

[0103] METHODS:

[0104] (i) Eotaxin was a pool of both peaks of bioactivity (fractions51+52 and fraction 54) from the final reversed phase HPLC separationdescribed in Example 2 (see FIGS. 3, 4 and 5). ¹¹¹In-eosinophilaccumulation in guinea-pig skin was measured over 4 h as described inExample 1 (see FIGS. 1 and 2). In the same animals (n=4) additionalsites were injected with eotaxin 30 minutes before killing.

[0105] (ii) For the binding studies, 4×10⁵ eosinophils were incubatedwith 0.1 nM ¹²⁵I-RANTES and various concentrations of cold ligand (50 μlat 0° C. for 2 h). The Hank's buffered salt solution contained 30 mMHEPES, 10 mM EDTA, 0.1% sodium azide and 1% BSA at pH 7.5. Results arethe mean of two assays each done in triplicate.

[0106] (iii) For measurement of intracellular calcium levels human andguinea-pig eosinophils (10⁷ cells/ml in Ca²⁺/Mg²⁺-free PBS+0.1% BSA)were loaded with fura-2-acetoxymethyl ester (2.5 μM, 30 min at 37° C.).After two washes cells were resuspended at 10⁶ cells/ml inCa²⁺/Mg²⁺-free PBS containing 10 mM HEPES, 0.25% BSA and 10 mM glucose(pH 7.4). Aliquots were dispensed into quartz cuvettes and the external[Ca²⁺] adjusted to 1 mM with CaCl₂. Changes in fluorescence weremonitored at 37° C. using a Perkin Elmer LS50 spectrophotometer atexcitation wavelengths 340 nm and 380 nm and emission wavelength 510 nm.[Ca²⁺]_(i) levels were calculated as described previously (34) using theratio of the two fluorescence readings and a Kd for Ca²⁺ binding at 37°C. of 224 nM. Peripheral human eosinophils were prepared as describedpreviously (35) by density centrifugation on Percoll followed byimmunomagnetic removal of CD16⁺ neutrophils using the MACS system.Guinea-pig eosinophils were prepared as described in Example 1 (11,12).

[0107] (iv) To test for suppression of eosinophil accumulation in vivousing human RANTES, accumulation of ¹¹¹-labelled guinea-pig eosinophilsin skin sites was measured as described above. Guinea-pigs were injectedwith 1.8 pmol eotaxin (n=2), 100 pmol RANTES (n=1) or both 1.8 pmoleotaxin and 100 pmol RANTES (n=2). Intradermal saline was used ascontrol.

[0108] (v) For testing of receptor antagonist activity, thefura-2-acetoxymethyl ester-loaded guinea-pig eosinophils were stimulatedwith 3 nM eotaxin with or without pretreatment with human RANTES.

[0109] (vi) To investigate the effect of aerosol exposure of guinea-pigsto eotaxin, naive guinea-pigs were exposed to an aerosol of eithereotaxin or a control medium (n=8 per group). Exposure was performed byplacing two animals in a single chamber and nebulising 24 pmol ofectaxin dissolved in 10 ml PBS containing BSA carrier protein at 80μg/ml over a period of 35-40 minutes. Control animals receivedaerosolised PBS/BSA in the same manner. The animals were killed 20 hoursafter exposure to eotaxin or control medium. BAL fluid (5×10 ml HBSS, 10mM EDTA, pH 7.35) was recovered and centrifuged (300 g, 20 min, 4° C.).Total BAL cell count was determined by haemocytometer and differentialcell counts performed on stained (DiffQuick) cytospin preparations (3per animal, 400 cell counts per slide).

[0110] RESULTS:

[0111] The results are presented in FIGS. 10, 11, 12, 13, 14, 15, 16, 17and 18.

[0112]FIG. 10 shows that guinea-pig eotaxin (1.6 pmol) inducessignificant ¹¹¹In-eosinophil accumulation in vivo 30 min (open squares,p<0.01) and 4 h (filled squares, p<0.01) after intradermal injection. Incontrast, the recombinant human proteins, RANTES, MIP-1α and MCP-1, atdoses up to 100 pmol, were without effect over 4 hours. FIG. 11 insetshows that eotaxin and RANTES, but not the C—X—C chemokine IL-8, inhibitthe binding of ¹²⁵I-RANTES (B₀=14.4%) to guinea-pig eosinophils invitro. FIG. 17 shows (a) that human RANTES does not induce significanteosinophil accumulation and (b) that human RANTES inhibits to asubstantial degree the eosinophil accumulation induced by eotaxin, whichsuggests that ANTES acts as a receptor antagonist for eotaxin in vivo

[0113]FIG. 12 shows that eotaxin, RANTES and, at high concentrationonly, MCP-1 induce elevation of intracellular calcium levels in humaneosinophils in vitro. Traces are with eosinophils from one donor. In twoother donors 2 nM eotaxin gave a mean calcium elevation of 61 nM. In thethree donors (97.3±2% eosinophils) responses to 10 nM RANTES were 194±74nM [Ca²⁺]_(i) and responses to 100 nM MCP-1 were 93±38 nM [Ca²⁺]_(i).

[0114]FIG. 13 shows that guinea-pig eotaxin, but not human RANTES orMCP-1, elevates intracellular calcium levels in guinea-pig eosinophilsin vitro. Traces are with cells from one donor. Tn three donors(97.5±0.8% eosinophils) responses were: 2 nM eotaxin, 90±13 nM[Ca²⁺]_(i); 100 nM RANTES, 2.0±1.7 nM [Ca²⁺]_(i); 100 nM MCP-1, 3.3±0.7nM [Ca²⁺]_(i).

[0115]FIG. 14 and the inset FIG. 15 show that prior treatment ofguinea-pig eosinophils with RANTES (100 nM) inhibits the increase inintracellular free calcium levels seen when eosinophils are treated witheotaxin (3 nM) alone. RANTES appears to be acting as a receptorantagonist in guinea-pig eosinophils. FIG. 16 is a dose-response curveusing 3 nM eotaxin and increasing amounts of RANTES.

[0116]FIG. 18 shows that guinea-pig eotaxin, administered as an aerosol,induces eosinophil accumulation in guinea-pig airways in vivo, whereassubstantially no accumulation of neutrophils is observed.

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[0149] 34. Grynkiewicz, G., Poenie, M. & Tsien, R. Y. J. Biol. Chem.

1 11 1 73 PRT Cavia porcellus VARIANT (54)..(55) Unknown or other 1 HisPro Gly Ile Pro Ser Ala Cys Cys Phe Arg Val Thr Asn Lys Lys 1 5 10 15Ile Ser Phe Gln Arg Leu Lys Ser Tyr Lys Ile Ile Thr Ser Ser Lys 20 25 30Cys Pro Gln Thr Ala Ile Val Phe Glu Ile Lys Pro Asp Lys Met Ile 35 40 45Cys Ala Asp Pro Lys Xaa Xaa Trp Val Gln Asp Ala Lys Lys Tyr Leu 50 55 60Asp Gln Ile Ser Gln Xaa Thr Lys Pro 65 70 2 73 PRT Cavia cobaya 2 HisPro Gly Ile Pro Ser Ala Cys Cys Phe Arg Val Thr Asn Lys Lys 1 5 10 15Ile Ser Phe Gln Arg Leu Lys Ser Tyr Lys Ile Ile Thr Ser Ser Lys 20 25 30Cys Pro Gln Thr Ala Ile Val Phe Glu Ile Lys Pro Asp Lys Met Ile 35 40 45Cys Ala Asp Pro Lys Lys Lys Trp Val Gln Asp Ala Lys Lys Tyr Leu 50 55 60Asp Gln Ile Ser Gln Thr Thr Lys Pro 65 70 3 24 DNA Artificial SequenceDescription of Artificial Sequencehypothetical 3 tgctgtttcc gngtnacnaacaaa 24 4 21 DNA Artificial Sequence Description of ArtificialSequencehypothetical 4 catcttgtcn ggcttnattt c 21 5 76 PRT human 5 GlnPro Asp Ala Ile Asn Ala Pro Val Thr Cys Cys Tyr Asn Phe Thr 1 5 10 15Asn Arg Lys Ile Ser Val Gln Arg Leu Ala Ser Tyr Arg Arg Ile Thr 20 25 30Ser Ser Lys Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Ile Val Ala 35 40 45Lys Glu Ile Cys Ala Asp Pro Lys Gln Lys Trp Val Gln Asp Ser Met 50 55 60Asp His Leu Asp Lys Gln Thr Gln Thr Pro Lys Thr 65 70 75 6 74 PRT human6 Asp Ser Val Ser Ile Pro Ile Thr Cys Cys Phe Asn Val Ile Asn Arg 1 5 1015 Lys Ile Pro Ile Gln Arg Leu Glu Ser Tyr Thr Arg Ile Thr Asn Ile 20 2530 Gln Cys Pro Lys Glu Ala Val Ile Phe Lys Thr Lys Arg Gly Lys Glu 35 4045 Val Cys Ala Asp Pro Lys Glu Arg Trp Val Arg Asp Ser Met Lys His 50 5560 Leu Asp Gln Ile Phe Gln Asn Leu Lys Pro 65 70 7 67 PRT human 7 LysSer Thr Thr Cys Cys Tyr Arg Phe Ile Asn Lys Lys Ile Pro Lys 1 5 10 15Gln Arg Leu Glu Ser Tyr Arg Arg Thr Thr Ser Ser His Cys Pro Arg 20 25 30Glu Ala Val Ile Phe Lys Asp Lys Glu Ile Cys Ala Asp Pro Thr Gln 35 40 45Lys Trp Val Gln Asp Phe Met Lys His Leu Asp Lys Lys Thr Gln Thr 50 55 60Pro Lys Leu 65 8 71 PRT guinea pig 8 Gly Val Asn Thr Pro Thr Cys Cys TyrThr Phe Asn Lys Gln Ile Pro 1 5 10 15 Leu Lys Arg Val Lys Gly Tyr GluArg Ile Thr Ser Ser Arg Cys Pro 20 25 30 Gln Glu Ala Val Ile Phe Arg ThrLeu Lys Asn Lys Glu Val Cys Ala 35 40 45 Asp Pro Thr Gln Lys Trp Val GlnAsp Tyr Ile Ala Lys Ile Asp Gln 50 55 60 Arg Thr Gln Gln Lys Gln Asn 6570 9 69 PRT human 9 Ser Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe SerTyr Thr Ser 1 5 10 15 Arg Gln Ile Pro Gln Asn Phe Ile Ala Asp Tyr PheGlu Thr Ser Ser 20 25 30 Gln Cys Ser Lys Pro Gly Val Ile Phe Leu Thr LysArg Ser Arg Gln 35 40 45 Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gln LysTyr Val Ser Asp 50 55 60 Leu Glu Leu Ser Ala 65 10 68 PRT human 10 ProMet Gly Ser Asp Pro Pro Thr Ala Cys Cys Phe Ser Tyr Thr Ala 1 5 10 15Arg Lys Leu Pro Arg Asn Phe Val Val Asp Tyr Tyr Glu Thr Ser Ser 20 25 30Leu Cys Ser Gln Pro Ala Val Val Phe Gln Thr Lys Arg Ser Lys Gln 35 40 45Val Cys Ala Asp Pro Ser Glu Ser Trp Val Gln Glu Tyr Val Tyr Asp 50 55 60Leu Glu Leu Asn 65 11 68 PRT human 11 Ser Pro Tyr Ser Ser Asp Thr ThrPro Cys Cys Phe Ala Tyr Ile Ala 1 5 10 15 Arg Pro Leu Pro Arg Ala HisIle Lys Glu Tyr Phe Tyr Thr Ser Gly 20 25 30 Lys Cys Ser Asn Pro Ala ValVal Phe Val Thr Arg Lys Asn Arg Gln 35 40 45 Val Cys Ala Asn Pro Glu LysLys Trp Val Arg Glu Tyr Ile Asn Ser 50 55 60 Leu Glu Met Ser 65

1. A substantially isolated chemoattractant protein capable ofattracting eosinophils and of inducing eosinophil accumulation and/oractivation in vitro and in vivo.
 2. A chemoattractant protein as claimedin claim 1, obtainable from a human or nonhuman animal after an allergenchallenge or other provoking stimulus.
 3. A chemoattractant protein asclaimed in claim 2, obtainable from bronchoalveolar lavage fluid of asubject after allergen challenge or other provoking stimulus.
 4. Achemoattractant protein as claimed in claim 1, obtainable from aninflammatory exudate fluid, or from an in vitro culture of macrophages,lymphocytes, neutrophils, mast cells, airway cells, connective tissuecells, vascular endothelial cells or eosinophils.
 5. A chemoattractantprotein as claimed in any one of claims 1 to 4, having a molecularweight in the range of from 6-16 kDa after purification on HLPC.
 6. Achemoattractant protein as claimed in claim 1, consisting of orcomprising an amino acid sequence as set out in SEQ.ID. NO. 1 or SEQ.ID.NO.
 2. 7. A chemoattractant protein as claimed in claim 1, consisting ofor comprising an amino acid sequence having homology with the amino acidsequence set out in SEQ.ID. NO. 1 or SEQ.ID. NO.
 2. 8. A chemoattractantprotein as claimed in claim 7, having at least 40% homology with theamino acid sequence set out in SEQ.ID. NO.
 1. 9. A chemoattractantprotein capable of attracting eosinophils and of inducing eosinophilaccumulation and/or activation in vitro and in vivo, which is apolypeptide or peptide fragment of a chemoattractant protein as claimedin any one of claims 2 to
 8. 10. A process for the production of achemoattractant protein as claimed in claim 1, which comprises obtainingbronchoalveolar lavage fluid or an inflammatory exudate from a human ora non-human animal challenged with a provoking stimulus, and isolating afraction that demonstrates eosinophil chemoattractant activity in vitroand/or in vivo.
 11. A process for the production of a chemoattractantprotein as claimed in claim 1, which comprises culturing in vitromacrophages, lymphocytes, neutrophils, mast cells, airway epithelialcells, connective tissue cells, vascular endothelial cells oreosinophils obtained from a human or a non-human animal, and isolatingfrom the cells or from the cell culture fluid a fraction thatdemonstrates eosinophil chemoattractant activity in vitro and/or invivo.
 12. A chemoattractant protein as claimed in any one of claims 1 to9, obtained by chemical synthesis or by recombinant DNA technology. 13.An agent that inhibits or otherwise hinders the production, release oraction of a chemoattractant protein as claimed in any one of claims 1 to9.
 14. An agent as claimed in claim 13, which is a receptor for achemoattractant protein as claimed in any one of claims 1 to 9, anantagonist for a chemoattractant protein as claimed in any one of claims1 to 9 at a receptor for that protein, or an agent that inhibits anagonist that binds to or activates a receptor for a chemoattractantprotein as claimed in any one of claims 1 to
 9. 15. An agent as claimedin claim 14, which is a receptor for the chemoattractant protein.
 16. Anagent as claimed in claim 14, wherein a receptor antagonist or anagonist inhibitor is a polypeptide in which the sequence of afull-length naturally-occurring chemoattractant protein as claimed inany one of claims 1 to 9 has been modified by amino acid substitution,or is a polypeptide or peptide comprising part of the amino acidsequence of a naturally-occurring chemoattractant protein as claimed inany one of claims 1 to 9, or is a polypeptide or peptide fragmentcomprising part of the amino acid sequence of a naturally-occurringchemoattractant protein as claimed in any one of claims 1 to 9, whichsequence has been modified by amino acid substitution.
 17. An agent asclaimed in claim 14, wherein a receptor antagonist is a chemotacticcytokine that binds to the same receptor as does the chemoattractantprotein.
 18. An agent as claimed in claim 13, which is an antibody. 19.An agent as claimed in any one of claims 13 to 18, for use as amedicament.
 20. Use of an agent as claimed in any one of claims 13 to 18in the manufacture of a medicament for the treatment of asthma oranother inflammatory disease.
 21. A pharmaceutical preparation whichcomprises, as active ingredient, an agent that inhibits or otherwisehinders the production, release or action of a chemoattractant proteinas claimed in any one of claims 1 to 9, in admixture or conjunction witha pharmaceutically suitable carrier.
 22. A pharmaceutical preparation asclaimed in claim 21, wherein the active ingredient is an agent asclaimed in any one of claims 14 to
 18. 23. A method of treating asthmaand other inflammatory diseases, comprising the administration of aneffective amount of an agent that inhibits or otherwise hinders theproduction, release or action of a chemoattractant protein as claimed inany one of claims 1 to
 9. 24. A method as claimed in claim 23, whereinthe agent is as claimed in any one of claims 14 to
 18. 25. Animmunoassay for an antigen or other assay for one member of a specificbinding pair, characterised in that the antigen or member of thespecific binding pair is a chemoattractant protein as claimed in any oneof claims 1 to
 9. 26. An immunoassay for an antibody, characterised inthat the antibody is an antibody that forms a complex with achemoattractant protein as claimed in any one of claims 1 to
 9. 27. Amethod of testing a compound for an inhibitory effect on the activity ofa chemoattractant cytokine in vitro, characterised in that thechemoattractant cytokine is a chemoattractant protein as claimed in anyone of claims 1 to
 8. 28. A method of determining the ability of asubstance to induce eosinophil accumulation and/or activation in vivo,which comprises administering the substance to a test animal previouslytreated with labelled eosinophils and subsequently determining thenumber of labelled eosinophils at a skin site.
 29. A method fordetermining the ability of a substance to inhibit eosinophilaccumulation and/or activation induced in vivo by an eotaxin wherein toan animal pretreated with labelled eosinophils is administered aneotaxin and the substance, and the number of labelled eosinophils at askin site are subsequently determined.